Determinação de cotinina urinária por HPLC-UV para avaliação da exposição de pacientes asmáticos ao tabagismo

Abstract

Although cigarette smoking is very old, concern about the harm caused by cigarette smoking is recent and has been reported in the literature for the first time in the 1970s. Cigarette smoke is a complex mixture of particulate matter and gas , In which more than 4000 substances have already been identified, and at least 50 of them have some carcinogenic potential. The nicotine present in the highest concentration in this smoke is the alkaloid responsible for the dependence. Smoking directly affects diseases of the respiratory tract, mainly individuals presenting with asthma, because besides aggravating the symptoms, it has a negative effect on the treatment. In this sense, the evaluation of the degree of exposure is necessary to estimate how this factor is influencing the treatment response of the asthmatic. Therefore, it is essential to biomonitor the individual to exposure to cigarette smoke, by determining the most stable nicotine metabolite and higher concentration, cotinine, eliminated by urine. The aim of this study was to determine the exposure levels of patients from the Nucleus Excellence of Asthma (NEA) by quantifying cotinine in urine. 1317 samples from patients with different degrees of asthmatic disease (control group, mild asthma and severe asthma) were analyzed. Cotinine levels were determined through the implantation and validation of a method already described in the literature that combines the use of an internal standard (IS), 2-phenylimidazole, extraction of IS and cotinine present in the urine with dichloromethane and determination by High pressure liquid chromatography (HPLC) with UV detection ( = 260 nm). The validation consisted of the evaluation of some parameters described by ANVISA resolution n ° 899 of May 29, 2003: linearity, selectivity, precision (intermediate and repeatability), accuracy, limits of detection and quantification. The method in question showed a good linearity in the working range (0-1500 μg.L-1), was selective to the analyte in question, besides presenting percentages of relative standard deviation (RSD) acceptable for the parameters intermediate precision and Repeatability (8.4 and 6.4%, respectively, for a cotinine urine standard of 100 μg.L-1). Accuracy was assessed by the percentage recovery of a standard of 500 μg.L-1 cotinine in urine, being found a value of 103.4%. The limits of detection and quantification were 6.46 and 19.59 μg.L-1, respectively. In general, NEA patients had low rates of exposure to cigarette smoke. About 86% of these had urinary cotinine concentrations below the limit of detection. In the comparison between the responses obtained by applying a standard questionnaire and the levels of cotinine that characterized the patient as an active smoker in the control, mild asthma and severe asthma patients, a contradiction percentage of 1.42, 0.73 and 6.0% respectively. This fact indicates that some patients with severe asthma omit their smoking status for fear of not being accepted into the program.